Modulation of line-1 reverse transcriptase

ABSTRACT

A reverse transcriptase encoded by L-1 (LINE-1) has been identified as a target molecule for treating or preventing cancers induced or mediated by this molecule. Method of treating or preventing such cancers in patients involves administration of a therapeutically effective amount of a composition having an inhibitor or antagonist of the reverse transcriptase in cells of the patients. The inhibitor or antagonist blocks lengthening of telomeres in telomerase negative cells. Methods and kits for detecting pathologically proliferating cells expressing L1RT are also disclosed.

This application claims the benefit of U.S. Provisional Application No.60/440,988 filed Jan. 15, 2003, and the text of application 60/440,988is incorporated by reference in its entirety herewith.

FIELD OF THE INVENTION

The present invention is directed to the field of cancer therapy.Specifically, target molecules have been identified modulation of whichregulates elongation of telomeres in telomerase negative cancerouscells. More particularly, it relates to the use of various inhibitorcompounds that interfere with human L1 (Line-1) retrotransposon encodedreverse transcriptase (L1RT) for treating or preventing L1RT inducedcancers. The invention also relates to screening methods for identifyingpharmacologically active compounds that may be useful for treatingL1RT-mediated proliferative diseases.

BACKGROUND OF THE INVENTION

An asymmetry in the synthesis of leading and lagging DNA strands createsthe “end problem” for replication of linear genomes⁸. To overcome this,eukaryotic chromosomes have specialized end structures, telomeres,consisting of TTAGGG repeats⁹. Telomerase is a ribonucleoprotein enzymethat elongates telomeres and therefore maintains chromosomal stabilityin majority of cancer cells during cell doubling. The gradual loss ofDNA from the ends of telomeres during cell doubling has been implicatedin the control of cellular proliferative potential in somatic cells¹⁰.

Normal cultured human cells have a limited replication potential inculture. As was first described by Hayflick, normal cells in culturereplicate until they reach a discrete point which population growthceases. This is termed M1 stage and is caused by the shortening of a fewtelomeres to a size that leads to a growth arrest called cellularsenescence. This stage can be bypassed in vitro by abrogation of thefunction of p53 and pRB human tumor suppressor genes. The cells then canproliferate until the telomeres have become critically shortened, whichproduces the M2 or crisis stage. The growth arrest in the M2 stage iscaused by balance between the cell proliferation and cell death rate. Atthis stage, when most of telomeres are extremely short, end-to-endfusions and chromosomal breakage-fusion cause marked chromosomalabnormalities and apoptosis. Under rare circumstances, a cell can escapeM2 and become immortal by stabilizing the length of its telomeres. Thisoccurs through the activation of the enzyme telomerase or an alternativemechanism of telomere lengthening (ALT).

Human germline² and the majority of cancer cells³ express telomerase.Telomerase is a ribonucleoprotein enzyme that elongates telomeres and,therefore, maintains chromosomal stability in majority of cancer cellsduring cell doubling. Indeed, elongation of shortened telomeres bytelomerase is a major mechanism of telomere maintenance in the humancancer cells. Inhibition of telomerase limits the growth of humantelomerase positive cancer cells¹¹ by decreasing telomere length, thesecompounds diminish the ability of these cancer cells to proliferate.Reverse transcriptase inhibitors have been used previously to treatcancer. In in vitro tests, tumor cells treated with the reversetranscriptase inhibitors underwent apoptosis after 14 days.

Elongation of shortened telomeres by telomerase is a well knownmechanism of telomere maintenance in the human cancer cells. However upto 30% of human tumors of different types do not express telomerase. Thepresence of ALT was reported in up to 30% of human tumors of differenttypes, tumor-derived cell lines and human cell lines immortalized invitro^(4,5,12,13), and up to 50% in some subsets of tumors andimmortalized cell lines¹⁴.

Currently, strategies aimed at selectively treating the cancers fromtelomerase positive cells involve modulation of TERT function or lengthof telomeres by antisense strategy, dominant negative mutants orpharmacological agents (see, Bisoffi et al., Eur J Cancer, 1998,34:1242-1249; Roth et al., Leukemia, 2003, 17:2410-2417; Damm et al.,EMBO J., 2001, 20:6958-6968; U.S. Pat. Nos. 6,294,332, 6,194,206,6,156,763 and 6,046,307). Selective modulation (i.e., selectiveinhibition or promotion) of telomerase negative cancer cells may also bemade possible if the target molecule(s) responsible for the lengtheningof telomeres in such cells are known. Thus, there is need foridentifying target molecules responsible for the lengthening oftelomeres in telomerase negative cells and identifying agents forselectively interfering with the identified target molecules so thathuman tumors of types that do not express telomerase may also beprevented or treated.

SUMMARY OF THE INVENTION

It has now been found that a product of L1 (Line-1) retrotransposonreverse transcriptase nucleic acid is associated with the lengtheningand therefore maintenance of telomeres in certain cancer cells.Specifically, it has been found that interference with the expression ofreverse transcriptase encoded by the L1 retrotransposon suppresses theelongation of telomeres in the cancer cells. More pecifically, it hasbeen found that interference with the expression of the L1 reversetranscriptase in telomerase negative cells leads to phenotypicmanifestations such as telomere shortening, cell cycle arrest andapoptosis or cell death. It is believed that the reverse transcriptaseis involved in maintaining telomeres probably by “slippage” mechanism oftelomeric DNA synthesis and/or telomere end targeted L1 transposonretrotransposition.

Still more specifically, it has been found that treatment of thetelomerase negative cells (ALT cells) with reverse transcriptaseinhibitor 3′-azido-2′,3′-dideoxythymidine (AZT) or suppression of L1reverse transciptase (L1RT) using antisense strategy induces progressivetelomere loss, G2 phase arrest, chromosomal abnormalities and eventualcell death.

Accordingly, in one embodiment of the invention, a method is providedfor treating tumors characterized by expression of L1RT and/or absenceof telomerase expression. Interference with L1RT expression or activitywill either directly result in cell death or will potentiate the effectsof chemotherapeutic agents that ultimately kill cells through apoptosis.In particular, the invention provides a method for inhibitingproliferation of L1RT expressing cells having potential for continuousincrease in cell number by administering inhibitors and antagonists ofL1RT. For example, L1RT expression can be suppressed or down regulatedby obtaining a DNA molecule having a cDNA sequence operably linked to apromoter such that it will be expressed in antisense orientation, thecDNA having all or part of the sequence of L1RT, and transfecting, withthe DNA molecule, the L1RT cells with potential for uncontrolledproliferation. The inhibitor or antagonist is optionally administeredwith a pharmaceutically acceptable carrier.

In another embodiment of the invention, a method for prevention of acancer in a person (e.g. a human) in need thereof is provided. Thecancer is due to the presence in the human of cells showing alternativelengthening of telomeres induced or mediated by L-1 (LINE-1)retrotransposon encoded reverse transcriptase in the cells of theperson. Lengthening of telomeres in cells induces a potential forcontinuous proliferation of such cells in the human body. The preventivemethod involves administration of a therapeutically effective amount ofa composition to the person. The composition has an inhibitor orantagonist of the reverse transcriptase. The inhibitor or antagonistblocks lengthening of telomeres in telomerase negative cells therebyinhibiting proliferation of L1RT expressing cells having potential forcontinuous increase in cell number. Preferably the inhibitor is one ormore nucleoside analogs, or a pharmaceutically acceptable salt of suchanalogs. A liquid or solid food material is enriched with inhibitor orantagonist. The food product can be, for example, a functional food inthe form of butter, margarine, biscuits, bread, cake, candy,confectionery, yogurt or another fermented milk product, or cerealsuitable for consumption by humans. Alterantively, it can be anutritional supplement, a nutrient, a pharmafood, a nutraceutical, ahealth food and/or a designer food. Periodically, the human is testedfor the presence of ALT cells. The use of inhibitor or antagonist may bestopped once the ALT cells are no longer detected.

In another embodiment of the invention, a method is provided forscreening candidate drugs or compounds to select drugs with potentialfor decreasing the rate of accumulation of tumor cells by incubating ortreating cells expressing L1RT with a candidate drug and monitoring oneor more desired biological effects the candidate drug(s) may have on thecells. If the candidate drug causes a desired bilogical effect, then thedrug is selected. Particularly preferred biological effects in such ascreening include progressive telomere loss, G2 phase arrest,chromosomal abnormalities or cancer cell death. The biological effectsmay also include inhibition of proliferation of telomerase negativecells transformed with various oncogenes such as, for example, ras.

The invention further provides methods and kits for detectingpathologically proliferating cells expressing L1RT. These and otherembodiments of the invention will be described in more detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a dot blot of total cellular RNA from ALT and telomerasepositive cell lines with telomere specific probe. 1, U-2 OS. 2, Saos-2.3, no RNA. 4, HEC-1.5, HeLa.

FIG. 2 illustrates flow cytometry data showing decrease in telomerelength, massive apoptosis and changes in cell cycle after 14 days oftreatment of ALT cell lines with AZT. Telomere specific fluorescence inG2 phase of cell cycle, (a) Saos-2; (b) U-2 Cell cycle distribution²²(c) Saos-2; (d) U-2 OS. Untreated cells—dark, treated—grey.

FIG. 3 illustrates flow cytometry data showing changes in DNA synthesisrate, cell cycle distribution and telomere length in U-2 OS cellstreated with AZT for different amounts of time, a, b, c, d no treatmentand treatment for 10, 17, and 40 days respectively. Cell cycledistribution²⁴—left. Staining for BdU incorporation (FITC) andPI²⁴—middle. Staining with PNA-FITC and PI—right. The numbers indicatetelomere specific fluorescence measured in arbitrary units²² in G1 andG2 phases respectively.

FIG. 4 shows a schematic representation of L1 reverse transcriptaseantisense targeting strategy.

FIG. 5 illustrates flow cytometry data showing changes in cell cycledistribution and telomere length in U-2 OS cells transfected with L1targeted antisense construct, (a) no treatment; (b) sense construct; (c)antisense construct.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses that LINE-1 (L1) retrotransposon encodedreverse transcriptase (L1RT) enzyme is involved in lengthening oftelomeres in certain human cancer cells. Specifically, the presentinvention discloses that L1RT is involved in lengthening of telomeres incertain tumor tissues including telomerase negative tumors and thetumor-derived cell lines, and identifies L1RT enzyme or the sequencesencoding it as a target for controlling the proliferative properties ofthe tumor cells or inducing apoptosis of these cells.

The telomerase negative tumors and the tumor-derived cell lines arethose that do not express or have the endogenous telomerase and yet showlengthening of telomeres, also referred to herein as alternativelengthening of telomeres (ALT). The L1RT mediated telomere lengtheningin cells can be characterized by the presence of long and heterogeneoustelomeres relative to the telomere lengthening mediated by telomerase.One skilled in the art would know how to determine the presence of longand heterogeneous telomeres characteristic of ALT in cells by carryingout, for example, TRF assay (see, Bryan et al., 1997, Nature Medicine,3:1271-1274).

L1 reverse transcriptase, which is encoded by ORF2 of L1retrotransposon, has already been characterized and its nucleic acid andprotein sequences are known in the art (GeneBank GI: 5070620; Ostertaget al., 2000, Determination of L1 retrotransposition kinetics incultured cells, Nucleic Acids Res. 28, 1418-1423; Kimberland et al.,1999, Full-length human L1 insertions retain the capacity for highfrequency retrotransposition in cultured cells, Hum. Mol. Genet. 8 (8),1557-1560). In the present invention, it has been discovered that L1RTadds telomeric DNA repeats to chromosomes in telomerase negative cells.

Accordingly, in an aspect of the present invention, methods forpreventing or treating disorders caused by the presence ofinappropriately or pathologically proliferating cells or immortal cellsin animals are provided. The inappropriately or pathologicallyproliferating cells or immortal cells exist and reproduce independentlyof cells' normal regulatory mechanisms. These cells are pathologicbecause they deviate from normal cells as a result of activity of acellular element, i.e., L1RT. Of course, the inappropriatelyproliferating cells as used herein may be benign hyperproliferatingcells but unless stated otherwise these cells refer to malignanthyperproliferating cells such as cancer cells characteristic of, forexample, osteosarcoma, breast carcinoma, ovarian carcinoma, lungcarcinoma, adrenocortical carcinoma or melanoma.

In particular, methods for preventing or treating human tumorscharacterized as expressing L1RT are provided. The prevention ortreatment of the disorders, according to the present invention, areachieved by the utilization of inhibitors or antagonists of L1RT. Theinhibitor(s) or antagonist(s) used in the present invention are thosethat directly or indirectly interact with L1RT to inhibit its expression(or activity) and/or those that get incorporated into telomere and thusprevent telomere from further elongation despite the functional L1RTthereby inhibiting the growth of cells expressing L1RT. Thus, theinhibitors or antagonists of L1RT are used for inhibiting the growth ofcells. For example, when the inhibitors or antagonists of L1RT areadministered to a patient, these cause progressive telomere shortening,cell cycle arrest in the cells and/or massive apoptosis of the cellsexpressing L1RT. In the present invention, inhibiting the growth mayalso mean reducing or preventing cell division. Inhibition of growth ofcells expressing L1RT, in the present invention, may be about 100% orless but not 0%. For example, the inhibition may be from about 10% toabout 100%, preferably at least about 25%, and more preferably at leastabout 50%, still more preferably at least about 90%, 95% or exactly 100%compared to that of the control cells (control cells express L1RT butare not treated with an inhibitor or antagonist). The inhibition ofgrowth can be measured by any methods known in the art. For example,viable cell number in treated samples can be compared with viable cellnumber in control samples, determined after incubation with vitalstains. In addition, growth inhibition can be measured by assays thatcan detect reductions in cell proliferation in vitro or in vivo, such astritiated hydrogen incorporation assays, BdU incorporation assay, MTTassay, changes in ability to form foci, anchorage dependence or losingimmortalization, losing tumor specific markers, and/or inability to formor suppress tumors when injected into animal hosts (Dorafshar et al.,2003, J Surg Res., 114:179-186; Yang et al., 2004, Acta Pharmacol Sin.,25:68-75).

The development of a cancerous tumor from a single immortalized cell orfew such cells may take several months to years in humans. By practisingthe present invention, however, cancer can be prevented because theability of the tumorigenic ALT cells treated with L1RT inhibitors losetheir proliferative potential before they have had a chance to grow intoa tumor. Further, periodic preventative administration of L1RTinhibitors or antagonists to at risk groups in order to stop tumorprogression before clinical manifestation of cancer could potentiallydecrease the rate of new cancer cases significantly.

The inhibitor or antagonist of the L1RT used in the present inventioncan be an inorganic compound, an organic compound, an antisensesequence, a double-stranded RNA (dsRNA) corresponding to a definedtarget region in L1RT mRNA, a dominant negative mutant of the L1RTprotein, an antibody or a small molecule.

In one embodiment of the invention, organic compounds such as, forexample, nucleoside analogs are used as inhibitors or antagonists ofL1RT. Thus, one of the approaches for targeting L1RT is byadministration of nucleoside analog(s) to cancer patients. Thenucleoside analogs can mimic the building blocks used by L1RT to extendthe chromosomal ends in telomerase negative cells. These fake buildingblocks (i.e., nucleoside analogs) that are incorporated into chromosomalends by L1RT may interfere with the function of the telomeres andthereby contributing to telomere shortening, cell cycle arrest and celldeath.

There are a number of nucleoside analogs known to one skilled in theart. Indeed, nucleoside analogues are known class of antiretrovirals anda number of nucleoside analog drugs have been approved for the treatmentof HIV infected humans. These drugs do stop HIV from multiplying byinterfering with copying HIV's genetic material (RNA) into the form ofDNA. Examples of nucleoside analogues that may be used in the presentinvention are, 3′-azido-2′3′-dideoxythymidine (AZT),2′,3′-dideoxyinosine (ddI) and 2′,3′-didehydro-3′-deoxythymidine(d4T).The other known nucleoside analogues such as Dideoxycytidine (ddC) and3TC may be used in some situations as determined by one skilled in theart.

Since L1RT is a key factor in cancers of telomerase negative cells, thepresent discovery of noncompetitive inhibitors of the activity of thiskey enzyme represents a potential breakthrough in cancer research andtreatment. The demonstration that nucleoside analogs (e.g., AZT) clearlyblock ALT cancer in a widely accepted model systems (described below),confirms that the present invention truly represents a dramaticbreakthrough. Although not suggesting the advantageous uses madepossible by this invention, the previous administration of AZT to AIDSpatients means that AZT can be readily administered to cancer patients.

Indeed, nucleoside analogs have been used to modify telomerase activityin cancer cells to levels close to that found in normal cells as a meansfor cancer therapy. The concentration of nucleoside analogs required toinhibit L1RT, however, can be several fold lower than that required toinhibit telomerase. For example, the concentration of AZT required forinhibiting L1RT activity can be orders of magnitude lower (e.g., 10 to1000 fold lower) than that required for inhibiting telomerase activity.The susceptibility of L1RT to such low levels of nucleoside analogs isquite unexpected and this unexpected finding now offers an advantageousavenue of therapy for treatment of L1RT specific cancers. Importantly,the present invention provides for the selection of effective dosessignificantly lower than the levels that may otherwise be used in cancerpatients. The studies of this invention indicate that AZT will be usefulin cancer at levels that achieve nanomolar drug levels rather than 200μM to 800 μM.

Further, the present use of nucleoside analogs to AIDS patients, coupledwith the ability to use significantly lower doses of AZT for HIVtherapy, should speed regulatory approval for the use of AZT in thetreatment of L1RT induced and/or mediated cancers. Moreover, thisinvention is not limited to the use of AZT to treat L1RT induced and/ormediated cancers. In fact, the use of L1RT inhibitors is broadlyapplicable to a range of other disorders in which L1RT is a factor.These include, for example, L1 induced mutations in the gene for bloodfactor VIII inducing hemophilia A, in the X-linked retinitis pigmentosa2, in the dystrophin gene, in the DMD gene resulting in X-linked dilatedcardiomyopathy and in the X-linked gene CYBB causing chronicgranulomatous disease (Woods-Samuels et al., 1989, Genomics, 4:290-296;Schwahn et al., 1998, Nat. Genet., 19:327-332; Holmes et al., 1994, NatGenet., 7:143-148; Yoshida et al., 1998, Hum Mol Genet., 7:1129-1132;Brouha et al., 2002, Am J Hum Genet., 71:327-336).

The nucleoside compounds may be administered either singly or incombinations of different analogs and by any routes of administration,including oral administration. AZT is a preferred nucleoside analog. AZTis commercially available and AZT formulations are described in a numberof U.S. patents. See, for example, U.S. Pat. No. 5,683,990. The cellswith ALT will be selectively targeted because these cells depend on L1RTfor elongating or maintaining telomeres and the elongation ormaintenance of telomeres requires the incorporation of the nuclosidesand/or their analogs. To the extent any specific targeting agent isdesired for delivering the analogs to exert anti-cancer effects, the useof targeted AZT and/or other analogs are contemplated herein.Accordingly, in some embodiments, pharmaceutical compositions may havethe active compound, in this case, AZT or a other nucleoside analog,which has been conjugated to a targeting agent (e.g., a peptide) forspecific delivery to particular target cells or to nuclear portionwithin cells.

In another aspect of the invention antisense sequence(s), also referredto herein as antisense oligonucleotide(s) or antisense polynucleotide(s)are used as inhibitors or antagonists of L1RT. The antisense sequencesin the present invention are either substantially or fully complementaryto a nucleic acid encoding L1RT. The complementarity (whether full orsubstantial complementarity) of the antisense sequences is such thatthey specifically hybridize with the target nucleic acid sequence andinterfere with L1RT function, expression or otherwise, and theinterference is sufficient to inhibit the growth of the cells.

The nucleic acid encoding L1RT can be DNA, RNA transcribed from such DNAor a cDNA of the RNA. The L1 nucleic acid and amino acid sequences ofvarious mammals, such as mouse, monkey and humans have been sequenced(see GenBank Accession numbers AY053456, AF036235, AF148856 andGI5070620) (see also, GenBank protein accession AAD39215 for L1RT ORF2sequence). In the context of the present invention, L1RT mRNA is apreferred nucleic acid for which antisense nucleic acid sequences aredesigned. For example, a series of antisense phosphorothioateoligonucleotides, 20 or more nucleotides in length, targeting thenucleic acid encoding L1RT are designed. Generally, the antisensesequences in the present invention may be designed to bind to thepromoter or other control regions and coding and/or non-coding regionsof L1RT. The antisense sequences preferably target L1RT nucleic acidsequence portion encompassing a start codon. It is also contemplatedthat the most effective antisense sequences or constructs will includeregions complementary to coding and non-coding regions of L1RT. One canreadily test the effectiveness of a given antisense construct simply bytesting the construct in vitro to determine whether normal cellularfunction is affected. It is preferred that the selected antisensesequence inhibits L1RT activity or expression to the level that isinsufficient for inducing or mediating telomere lengthening in ALTcells.

Interference with L1RT expression can happen due to any mechanism. Forexample, it is believed that such antisense sequences bind to, andinterfere with the translation of, the sense L1RT mRNA. Alternatively,the antisense sequence may render the L1RT mRNA susceptible to nucleaseor ribozyme digestion, interfere with transcription, or interfere withprocessing of L1RT mRNA, repress transcription of mRNA from the L1RTgene, or act through some other mechanism, e.g., through ribozymes.Ribozymes, which are well known to those skilled in the art, aremolecules of RNA that have catalytic activity. The ribozymes of theinvention are antisense sequences that bind and enzymatically cleave andinactivate L1RT RNA. Useful ribozymes can comprise 5′- and 3′-terminalsequences complementary to the L1RT RNA and can be engineered by one ofskill on the basis of the L1RT RNA sequence. However, the particularmechanism by which the antisense sequences interfere with L1RTexpression is not critical so long as the end result is met.

Generally, to assure specific hybridization, the antisense sequence issubstantially complementary to the target L1RT mRNA sequence. In certainembodiments, an antisense sequence that is fully or exactlycomplementary to the target nucleic acid sequence or two or moreantisense sequences fully complementary to different subsequences of agiven L1 RT target nucleic acid sequence may be used. A Subsequence is asequence of nucleic acid residues or nucleotides that is a part of alonger sequence of nucleic acid residues such as, for example, anantisense sequence corresponding to nucleotides 1987-2800 of human L1reprotransposon (GenBank GI: 5070620).

TABLE  Exemplary sequences for use in interfering with L1RT mRNA.SEQ ID NO: Nucleic Acid Sequence SEQ ID NO: 15′-atga caggatcaac ttcacacata acaatattaa (a sequencectttaaatat aaatggacta antisense to L1RTaattctgcaa ttaaaagaca cagactggca agttggataa mRNA results whenagagtcaaga cccatcagtg the sequence settgctgtattc aggaaaccca tctcacgtgc agagacacac forth herein, SEQ IDataggctcaa aataaaagga NO: 1, in reversetggaggaaga tctaccaagc caatggaaaa caaaaaaagg orientation, iscaggggttgc aatcctagtc expressed in antctgataaaa cagactttaa accaacaaag atcaaaagag expression vector)acaaagaagg ccattacata atggtaaagg gatcaattca acaagaggag ctaactatcctaaatattta tgcacccaat acaggagcac ccagattcat aaagcaagtc ctcagtgacctacaaagaga cttagactcc cacacattaa taatgggaga ctttaacacc ccactgtcaacattagacag atcaacgaga cagaaagtca acaaggatac ccaggaattg aactcagctctgcaccaagc agacctaata gacatctaca gaactctcca ccccaaatca acagaatatacatttttttc agcaccacac cacacctatt ccaaaattga ccacatagtt ggaagtaaagctctcctcag caaatgtaaa agaacagaaa ttataacaaa ctatctctca gaccacagtgcaatcaaact agaactcagg attaagaatc tcactcaaag ccgctcaact acatggaaactgaacaacct gctcctgaat gactactggg tacataacga aatgaaggca gaaataaagatgttctttga aaccaacgag aacaaagaca ccacatacca gaatctctgg gacgcattcaaagcagtgtg tagagggaaa tttatagcac taaatgccta caagagaaag cagga-3′SEQ ID NO: 2 (a 5′-CCA GAG ATT CTG GTA TGT GGT GTC TTT GTT-3′sequense antisense to a portion of L1RT mRNA) SEQ ID NO: 3 (a5′-CTT TCT CTT GTA GGC ATT TAG TGC TAT AAA-3′ sequense antisenseto a portion of L1RT mRNA) SEQ ID NO: 4 (a5′-CTC TTG CTT TTC TAG TTC TTT TAA TTG TGA-3′ sequense antisenseto a portion of L1RT mRNA) SEQ ID NO: 5 (a5′-CTT CAG TTC TGC TCT GAT TTT AGT TAT TTC-3′ sequense antisenseto a portion of L1RT mRNA) SEQ ID NO: 6 (a5′-TCC TGC TTT CTC TTG TAG GCA -3′ sequense antisenseto a portion of L1RT mRNA)

The antisense sequences, e.g., DNA, RNA, modified, analogues or the likecan be made using any suitable method for producing a nucleic acid, suchas the chemical synthesis and recombinant methods disclosed herein (see,examples section) or such methods known to one of skill in the art. Inone embodiment, for example, antisense RNA molecules of the inventionmay be prepared by de novo chemical synthesis or by cloning. Forexample, an antisense RNA that hybridizes to L1RT mRNA can be made byinserting (ligating) a sequence set forth in SEQ ID NO:1 in reverseorientation, operably linking it to a promoter and expressing it in anexpression vector (e.g., plasmid). Provided that the promoter and,preferably termination and polyadenylation signals, are properlypositioned, the strand of the inserted sequence corresponding to thenoncoding strand will be transcribed and act as an antisense sequence ofthe present invention.

In some embodiments, the antisense sequences may also include modifiedantisense nucleic acid sequences having nucleotide additions,substitutions, deletions or modifications, or other nucleic acidsequences or non-nucleic acid moieties so long as specific binding tothe relevant target sequence, i.e., L1RT RNA or its gene/cDNA, isretained as a functional property of the sequences.

For example, a modified antisense nucleic acid sequence consisting ofthe nucleotides identical to that set forth in SEQ ID NO: 2, 3, 4, 5 or6 except that, over the entire length corresponding to the nucleotidesequence of SEQ ID NO: 2, 3, 4, 5 or 6, the modified antisense nucleicacid sequence has one or more nucleotide substitutions, deletions orinsertions. Identity or similarity, as known in the art, is arelationship between two or more polynucleotide sequences as determinedby comparing the sequences. Identity also means the degree of sequencerelatedness between polynucleotide sequences, as determined by the matchbetween strings of such sequences from 5′ to 3′ end for polynucleotides.“Identity” can be readily calculated by art known methods. See e.g.,Altschul et al., Nucleic Acids Res., 25:3389-3402 (1997). For example,sequence identity may be optimized by alignment algorithms known in theart and calculating the percent difference between the nucleotidesequences. Effective antisense sequences can be determined by using, forexample, GCG (Genetics Computer Group, Madison Wis.) or combinatorialarrays of oligonucleotides or DNA microarrays, which techniques areknown to one skilled in the art.

In the present invention, L1RT antisense polynucleotides, RNA, DNA ormodified nucleic acid that can be produced by direct chemical synthesismay also be used. Chemical synthesis is generally preferred for theproduction of oligonucleotides or for oligonucleotides andpolynucleotides containing nonstandard nucleotides (e.g., probes,primers and antisense oligonucleotides) for use in the presentinvention. Direct chemical synthesis of nucleic acids can be carried outby procedures known in the art. One of ordinary skill in the art willrecognize that while chemical synthesis of DNA may often be limited tosequences of about 100 or 150 bases, longer sequences may be obtained bythe ligation of shorter sequences or by more elaborate syntheticmethods. It will be appreciated that the L1RT antisene oligonucleotidesof the invention can be made using nonstandard bases or nonstandardbackbone structures to provide desirable properties such as, forexample, increased nuclease-resistance, tighter-binding, stability or adesired T_(m)).

A wide variety of useful modified oligonucleotides may be produced,including peptide nucleic acid (PNA). Peptide nucleic acid is ananalogue of DNA in which the backbone is a pseudopeptide (an amide, inparticular N-ethylaminoglycine backbone) rather than a sugar (see, PeterE. Nielsen (Ed), Peptide Nucleic Acids: Protocols and Applications,First Edition, 1999, Horizon Scientific Press). Such a backbone has beenreported to result in stronger binding and greater specificity thannormally achieved. In addition, the unique chemical, physical andbiological properties of PNA have been exploited to produce powerfulbiomolecular tools, antisense and antigene agents, molecular probes andbiosensors. Further teaching of PNA compounds can be found in U.S. Pat.Nos. 5,539,082; 5,714,331 and 5,719,262.

In some embodiments, chimeric oligonucleotides, triplex-formingantisense sequences, RNA-DNA oligonucleotides (RDO), oligonucleotideshaving backbone analogues, such as phosphodiester, phosphorothioate,phosphorodithioate and such others known in the art may be synthesizedand used. For example, a series of antisense phosphorothioateoligonucleotides, 30 nucleotides in length, targeting a nucleic acidencoding L1RT may be used.

It is often useful to label the antisense polynucleotides of theinvention, for example, when the L1RT polynucleotides are to be used fordetection of L1RT expression, or for the diagnosis and prognosis ofconditions related to the inappropriate hyperproliferation. The labelsmay be incorporated by any of a number of means well known to those ofskill in the art. Suitable labels are any composition detectable byphotochemical, biochemical, immunochemical, chemical, or spectroscopicmeans. For example, useful labels include ³²P, ³⁵S, fluorescent dyes,enzymes (e.g., as commonly used in an ELISA), biotin-streptavadin,digoxigenin, haptens and proteins for which antisera or monoclonalantibodies are available, or nucleic acid molecules with a sequencecomplementary to a target. The label often generates a measurablesignal, such as radioactivity, that can be used to quantitate the amountof bound detectable moiety.

In another aspect of the invention double-stranded RNAs (dsRNAs)corresponding to a defined target region in L1RT mRNA, are used asinhibitors or antagonists of L1RT. The dsRNAs induce RNA-targetedgene-silencing of L1RT which result in reduction or loss of L1RTexpression in targeted cells. RNA-targeted gene silencing is well knownto one of ordinary skill in the art (Ahlquist, 2002, Science,296:1270-1273). The dsRNAs may be endogenously synthesized orexogenously applied but only catalytic amounts of dsRNA are required toinduce the silencing. A nucleotide sequence from a portion of the L1RTgene is chosen to produce inhibitory RNA, which may be partially orfully double-stranded type. The inhibition is specific because anucleotide sequence from a portion of the target gene is chosen toproduce inhibitory RNA.

There are different methods known in the art to induce RNA-targetedgene-silencing such as small interfering RNA, short hairpin RNA,expressed long interfering RNA, expressed short interfering RNA andexpressed short hairpin RNA. It is preferred that certain dsRNAs (smallinterfering RNA and short hairpin RNA) include modifications to eitherthe phosphate-sugar backbone or the nucleoside. The RNA duplex formationmay be initiated either inside or outside the cell. The RNA may beintroduced in an amount which allows delivery of at least one copy percell with higher doses of double-stranded material may yield moreeffective inhibition. Inhibition is sequence-specific in that L1RT mRNAnucleotide sequences corresponding to the duplex region of the RNA aretargeted for genetic inhibition. RNA sequences with insertions,deletions, and single point mutations relative to the target sequencecan also be found to be effective for inhibition.

The RNA may be delivered to cells or directly introduced intointercellular spaces of a tissue or into the vascular system of anorganism. It may also be delivered orally to the patients. Methods fororal introduction include direct mixing of dsRNA with food of thepatient, as well as engineered approaches in which a species that isused as food is engineered to express the RNA, then fed to the organismto be affected. Physical methods of introducing nucleic acids includeinjection directly into the cell or extracellular injection into thepatient of an RNA solution.

In another aspect, dominant negative mutants of the L1RT protein ornucleic acids having a sequence encoding a dominant negative mutant L1RTprotein or non-functional fragment or derivative thereof areadministered to inhibit L1RT function by interfering with theinteractions of L1RT and with other molecules in the cell. It isbelieved that the L1RT must directly interact with a portion of thetelomere for telomere eleongation.

Therefore, L1RT mutants that are defective in function but effective inbinding to the portion of the telomere can be used as a dominantnegative mutant to compete with the wild type L1RT. Dominantnon-functional L1RT can be engineered for expression in cancer cellsthat inappropriately overexpress L1RT. Given that the protein andnucleic acid sequences of the wild type L1RT is known, one skilled inthe art can create dominant negative mutants of L1RT suitable for use inthe present invention. Such dominant negative mutants may beadministered to cells in vivo or in vitro according to the standarddelivery methods already known in the art. In a preferred aspect of theinvention, the therapeutic nucleic acid has an L1RT nucleic acid that ispart of an expression vector that expresses a dominant non-functionalL1RT protein or fragment or chimeric protein thereof in cancer cells.

In another aspect of the present invention, antibodies or bindingportions thereof specific to L1RT are used as inhibitors or antagonistsof L1RT. Specifically, the present invention contemplates the preventionand treatment of L1RT induced cancer in humans as well as other animalsthrough the use of antibodies to L1RT. Both polyclonal and monoclonalantibodies and binding portions (Fab fragments and Fv fragments) of suchantibodies are contemplated in the context of the present invention.Such antibodies may be made in a variety of animals including, mice,rabbits, monkeys, chimpanzees, cows (e.g., in the milk) and birds. Thepresent invention also contemplates human and humanized antibodies. Theantibodies can be used preventively or during the acute stage ofpathological cell proliferation.

In one embodiment, the present invention contemplates a method in whichthe antibodies which bind to L1RT protein are administered so that theantibodies react with L1RT. In another embodiment, the antibodies arecombined with other reagents including but not limited to otherantibodies. The administration of antibodies can be carried out orally,parenterally or by other suitable routes.

The antibody production may be effected by techniques which arewell-known in the art. For example, mammalian lymphocytes are immunizedby in vivo immunization of the animal (e.g., a mouse) with human L1RTprotein or polypeptide. Such immunizations are repeated as necessary atintervals of up to several weeks to obtain a sufficient titer ofantibodies. Hybridomas may be produced and cultured, and the resultingcolonies are screened for the production of the desired monoclonalantibodies. Colonies producing such antibodies are cloned, and growneither in vivo or in vitro to produce large quantities of antibody.

The dose of a given inhibitor or antagonsit of L1RT can be determined byone of ordinary skill in the art upon conducting routine experiments.Prior to administration to patients, the efficacy may be shown inexperimental animal models. In this regard any animal model for L1RTinduced cancer known in the art can be used (Hahn et al., 1999, NatureMedicine, 5(10): 1164-1170; Yeager et al., 1999, Cancer Research,59(17): 4175-4179). For example, immunodeficient mice (Balb/c-ByJ-Hfh11nu; Jackson Laboratory) are obtained and maintained in pathogen-freeconditions prior to xenograft tumor induction. To create human ALT cellline that will be tumorigenic in mice the IIICF/c fibroblast cell line(showing ALT) is transfected with pSV2neo-EJras plasmid (containing theactivated c-Ha-ras oncogene from the EJ bladder carcinoma cell line)DNA, and selected with G418.

Xenograft tumors can be subcutaneously generated in immunodeficient miceby the injection of the transformed IIICF/c fibroblast cells. About2×106 cells may be injected subcutaneously into the mice, preferablyalong their dorsal flanks, anaesthetized with Metofane. The growingtumours may be measured every 2-3 days. Tumor growth was followed bymeasuring with a caliper the longest axis of the tumor and the axisperpendicular to this. Tumor volume may be calculated using the formula4/3πr³, where r is the radius of the tumor. The tumors may be excisedand weighed prior to processing. Tissues to be used for molecularbiological analysis may be snap frozen in liquid nitrogen and stored at−80.degree. C. The xenograft tumors will have no detectable telomeraseactivity in the Telomeric Repeat Amplification Protocol (TRAP) assay.The TRF length pattern diagnostic of cells showing ALT may be verifiedby Southern analysis.

After induction of tumors, mice in the experimental groups may betreated with AZT. Mice may be injected i.p. twice a day with solution ofAZT in PBS with a total daily dose of 10 mg/kg. Mice in control groupmay be injected with PBS. Alternatively, AZT at the same daily dose maybe given in drinking water. Mice in the control group will bear theactively growing tumors and none of the mice in experimental groups willhave tumors. As a separate set of controls, telomerase-positive tumorsin nude mouse may be induced by injecting the immunodeficient mice withWM1175 (malignant melanoma) or HUT292DM (lung cancer) cells instead ofthe tranformed IIICF/c fibroblast cells. The telomerase-positive tumorsin the immunodeficient mice cannot be inhibited by the AZT at the dosetested.

Thus, the efficacy of various inhibitors or antagonists may be shown instandard experimental animal models prior to administration to subjectsor patients. The subject, or patient, to be treated using the methods ofthe invention is preferably human, and can be a fetus, child, or adult.Other an animal mammals that may be treated can be mice, rats, rabbits,monkeys and pigs.

The inhibitors or antagonists can be used alone or in combination withother chemotherapeutics or otherwise. For example, therapy of L1RTinduced cancers may be combined with chemo and/or radiotherapy to treatcancers induced by telomerase or some other factors. Examples of chemotherapeutic agents known to one skilled in the art include, but are notlimited to, anticancer drugs such as bleomycin, mitomycin, nitrogenmustard, chlorambucil, 5-fluorouracil (5-FU), floxuridine (5-FUdR),methotrexate (MTX), colchicine and diethylstilbestrol (DES). To practicecombined therapy, one would simply administer to an animal an inhibitorcomponent of the present invention in combination with anotheranti-cancer agent in a manner effective to result in their combinedanti-cancer actions within the animal or patient. The agents wouldtherefore be provided in amounts effective and for periods of timeeffective to result in their combined presence in the region of targetcells. To achieve this goal, the agents may be administeredsimultaneously, either in a single composition, or as two distinctcompositions using different administration routes. Alternatively, thetwo treatments may precede, or follow, each other by, e.g., intervalsranging from minutes to hours or days. By way of example, and notlimitation, doses of AZT for systemic use may be 500 mg/kg per day forhuman adults, 20 mg/kg per day for mice and human infants.

Some variation in dosage may occur depending on the condition of thesubject being treated. The physician responsible for administration willbe able to determine the appropriate dose for the individual patient andmay depend on multiple factors, such as, the age, condition, filehistory, etc., of the patient in question.

Accordingly, the methods of the invention can be used in therapeuticapplications for conditions and diseases associated with L1RT inducedpathological proliferation of cells. Diseases that would benefit fromthe therapeutic applications of this invention include all diseasescharacterized by cell hyperproliferation including, for example, solidtumors and leukemias, and non-cancer conditions. It is furthercontemplated that the method of the invention can be used to inhibit thegrowth of cancer cells not only in an in vivo context but also in an exvivo situation. The method of the invention is particularly useful forinhibiting the growth of pathologically proliferating human cells exvivo, including, but not limited to, human cancer cells-osteosarcoma,breast carcinoma, ovarian carcinoma, lung carcinoma, adrenocorticalcarcinoma or melanoma.

The present invention provides methods and kits for identifyinginappropriately, pathologically or abnormally proliferating cells due tothe expression of L1RT in the cells. The methods can be used as ascreening method that aids in diagnosing the presence of a cancerouscell or tumor in a patient by determining the presence (and/or level) ofexpression of L1RT in tissue from the patient, the presence of L1RTexpression being indicative of cancer cells or pathological cellproliferation in the patient.

For example, cancerous tumor samples can be diagnosed by the detectionof L1 specific mRNA expression measured by a variety of methodsincluding, but not limited to, hybridization using nucleic acid,Northern blotting, in situ hybridization or RNA microarrays, or thepresence of L1 retrotransposon ORF1 and/or ORF2 encoded proteinsmeasured by variety of methods including, but not limited to, Westernblotting, immunoprecipitation or immunohistochemistry, or enzymaticactivity of reverse transcriptase.

Cancer cells showing ALT can also be diagnosed by determining theabsence of catalytic subunit mRNA expression (measured by a variety ofmethods including, but not limited to, Northern blotting, RNA protectionassay, in situ hybridization, RT-PCR, real time RT-PCR or RNAmicroarrays), or the absence of telomerase catalytic subunit translation(measured by a variety of methods including, but not limited to, Westernblotting, immunoprecipitation or immunohistochemistry). Anothercharacteristic of cells showing ALT is the presence of long andheterogeneous telomeres (Bryan et al., 1997, Nature Medicine,3:1271-1274). Accordingly, a diagnostic method may include detection ofthe presence of long and heterogeneous telomeres as an indicator ofcells with ALT. The method includes, but is not limited to, terminalrestriction digest and its modification, in situ hybridization with atelomere specific probe or flow cytometry with telomere specific DNA orPNA probes.

In a preferred embodiment, nucleic acid probes directed against L1RT canbe used to detect presence and/or increases in L1RT mRNA levels intissues undergoing rapid proliferation, such as primary cancer cells,including human osteosarcoma, breast carcinoma, ovarian carcinoma, lungcarcinoma, adrenocortical carcinoma or melanoma. Thus, the presentinvention provides methods of using nucleic acid probes that arecomplementary to a subsequence of an L1RT to detect and identifypathologically proliferating cells, including cancer cells. For example,the method for identifying a pathologically proliferating cell mayinvolve using a nucleic acid probe directed against an L1RT mRNA tocompare the level of expression of L1RT mRNA in a test cell with thelevel of expression of L1RT mRNA in a control cell. A test cell isidentified as a pathologically proliferating cell when the level of L1RTexpression is observed as in the control cell.

It is preferred that the nucleic acid probe used in the method of thepresent invention is fully complementary to a human L1RT nucleic acidsequence, preferably mRNA, and the test cell is a human cell. An exampleof nucleic acid probe that is fully complementary to a human L1RT RNAsequence is 5′-TCC TGC TTT CTC TTG TAG GCA-3′ (SEQ ID NO:6). The nucleicacid probe used in the method of the invention, however, may also besubstantially complementary to an L1RT mRNA or an L1RT retrotransposonRT sequence of human mouse or other mammal. It will be apparent to oneof ordinary skill in the art that substitutions may be made in thenucleic acid probe which will not affect the ability of the probe toeffectively detect the L1RT RNA in pathologically proliferating cells(e.g., cancer cells) and thus, such substitutions are within the scopeof the present invention. The nucleic acid probe used in the method ofthe present invention can be a DNA probe, or a modified probe such apeptide nucleic acid probe, a phosphorothioate probe, or a 2′-O methylprobe. The length of the nucleic acid probe may be from about 8 or 10 to50 nucleotides, preferably from about 15 to 25 in length. The method ofthe invention can be readily performed in a cell extract, cultured cell,or tissue sample from a human, a mammal, or other vertebrate.

The methods of the present invention are useful for detecting theinappropriately, pathologically or abnormally proliferating cells due tothe expression of L1RT in the cells in vitro, in cell cultures, and inhuman cells and tissues, such as solid tumors and cancers (e.g., humanosteosarcoma, breast carcinoma, ovarian carcinoma, lung carcinoma,adrenocortical carcinoma or melanoma).

The present invention also provides kits for detecting and/or inhibitinghyperproliferating cells or cancer cells. The kit can have a nucleicacid probe that is fully or substantially complementary to a subsequenceof an L1RT mRNA. The kits for inhibiting the proliferation ofpathologically proliferating cells, the kit comprising the step ofcontacting the cells with may have an agent, e.g., an antisenseoligonucleotide that is substantially complementary, preferably fullycomplementary, to a subsequence of an L1RT nucleic acid, which agentupon contacting the cells can affect pathological proliferation. Thekits can be in the form of a container containing one or more of theabove-discussed nucleic acid probes, antisense oligonucleotides, orother suitable agents with or without detection labels discussed herein.The kits may contain a suitable membrane for separation andhybridization of sample RNA, DNA or protein, preferably in the form ofan assay apparatus that is adapted to use with the claimed methods. Thekits can also include instruction manuals for carrying out the methodsof the present invention. The kits may also include reagents useful fordetecting the presence of the detectable labels and/or materials usefulin the performance of various assays including positive, negativecontrols, internal and/or external controls. Exemplary reagents andmaterials are RNA extraction buffers, hybridization buffers, test tubes,transfer pipettes, and the like.

The inhibitors or antagonists of the L1RT that can be used in methods ofthe present invention should not be limited in any way to the specificcompounds mentioned in the present application. Given that the presentinvention discloses a target responsible for hyperproliferation ofcells, a number of other useful inhibitors or antagonists of the L1RTcan be identified by simple screening methods. The active compounds mayinclude fragments or parts of naturally-occurring or prior artcompounds. However, prior to testing of such compounds in humans, it maybe necessary to test a variety of candidate agents in screening assaysto determine which have potential as anti-tumor drugs. A number ofassays are known in the art for determining the effect of a drug oncancer. Therefore, in particular embodiments, the present inventionconcerns a method for identifying or selecting compounds that willmodulate expression or activity of L1RT. Drugs which interfere with thebiological activity of L1RT are good candidates for anti-tumor drugs,because they affect one of the steps that leads to uncontrolledproliferation or a continuous increase in cell number.

Screening for compounds or drugs may be performed using purified L1RTenzyme, an in vitro model, cell cultures, a genetically altered cell oranimal, or xenograft model antitumor assays. Of particular interest arescreening assays for agents that have a low toxicity for human cells.Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, antisense polynucleic acids or small organiccompounds having a molecular weight of more than 50 and less than about2,500 daltons, analogs of purines and pyrimidines or combinationsthereof. Known pharmacological antitumor agents may be subjected tofurther chemical modifications, such as amidification, to producestructural analogs. If the screening assay is a binding assay, one ormore of the molecules may be joined to a label, where the label candirectly or indirectly provide a detectable signal. Various labels suchas radioisotopes, fluorochromes, chemiluminescent agents, enzymes andspecific binding molecules, particles, e.g. magnetic particles may beused. A variety other reagents like salts, neutral proteins, e.g.albumin, detergents, etc may also be used to facilitate optimalprotein-protein binding and/or reduce non-specific or backgroundinteractions. Reagents that improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents may beused.

For example, a screening assay or a method for identifying a compound oragent in its simplest form may include incubating a candidate compoundor compounds to be tested with a cell expressing L1RT under conditionsin which, but for the presence of the compound or compounds to betested, the interaction of L1RT and other cell components induces adetectable or measurable biological effect or a chemical effect (exampleaddition of nucleotides or analogs to the telomere or maintenance oftelomere length) and then determining the ability of L1RT to interactwith the cell components to induce the detectable or measurablebiological effect or the chemical effect in the presence of the compoundor compounds to be tested. If the candidate compound or the testedcompound modulates the interaction L1RT with other cell components, thenthat compound is selected.

Other assays of interest can be, for example, a cell line expressingL1RT or an expression construct having an L1RT gene may be introducedinto a cell line under conditions that allow L1RT expression. To thiscell line candidate agent(s) is(are) added, and the ability to inhibitor down-regulate L1RT activity is detected. The level of L1RT activitymay be determined by a functional readout or assay including alterationsin L1RT expression levels, binding or inhibition of binding to atelomere or some other substrate, apoptosis, presence or lack of growth,presence or lack of metastasis, presence or lack of cell division,presence or lack of cell migration, presence or lack of soft agar colonyformation, presence or lack of contact inhibition, presence or lack ofinvasiveness, and/or presence or lack of tumor progression or othermalignant phenotype.

For example, a method for determining the ability of a candidatecompound to decrease the wild-type L1RT expression in cells and toconcomitantly induce apoptosis in those cells may be carried out byobtaining a cell expressing L1RT, admixing a candidate substance withthe cell; and determining the ability of the candidate substance toreduce the L1RT content and/or telomere length on the chromosomes of thecell.

Another simple example to identify a candidate substance as beingcapable of interfering with L1RT expression can be as follows: one maymeasure or determine the L1RT status of a cell. If that cell has theability to express L1RT, its basal L1RT content in the absence of theadded candidate compound is measured. One may then add the candidatecompound to the cell and re-determine the wild-type L1RT expression inthe presence of the candidate compound. A candidate compound thatdecreases the L1RT expression relative to the cell's L1RT expression inthe absence of the test or candidate compound is indicative of acandidate compound with wild-type L1RT expression inhibiting capability.It can, therefore, have prophylactic and therapeutic cancer reducing andapoptotic potential.

The present invention also encompasses the use of various animal models.By developing or isolating cell lines that express L1RT one can generatedisease models in various laboratory animals. These models may employthe subcutaneous, orthotopic or systemic administration of cells tomimic various disease states. For example, the IIICF/c fibroblast cellline (ALT) can be transfected with pSV2neo-EJras plasmid DNA (containingthe activated c-Ha-ras oncogene from the EJ bladder carcinoma cellline), selected with G418, and injected subcutaneously into nude mice toobtain ALT tumors. The resulting tumors do not show any detectabletelomerase activity in telomeric repeat amplification protocol (TRAP)assay, and Southern analysis shows that they retained the TRF lengthpattern diagnostic of ALT (Yeager et al., Cancer Res. 1999,59(17):4175-9). Finally, telomerase knock out animals (e.g., telomeraseKO mice−/−; Rudolph et al., 1999, Cell, 96:701-712) or transgenicanimals that express a wild-type L1RT as a transgene in the animals maybe utilized as models for treatment. Of course, animal models provide auseful vehicle for testing combinations of agents as well. Determiningthe effectiveness of a compound in vivo may involve a variety ofdifferent criteria including, but are not limited to, survival, tumorregression, arrest or slowing of tumor progression, elimination oftumors and inhibition or prevention of metastasis.

Treatment of animals with test compounds will involve the administrationof the compound, in an appropriate form, to the animal. Administrationwill be by any route the could be utilized for clinical or non-clinicalpurposes, including but not limited to oral, nasal, buccal, rectal,vaginal or topical. Alternatively, administration may be byintratracheal instillation, bronchial instillation, intradermal,subcutaneous, intramuscular, intraperitoneal or intravenous injection.Specifically contemplated are systemic intravenous injection, regionaladministration via blood or lymph supply and intratumoral injection.

Of course, the screen may include appropriate control values (e.g., thelevel of L1RT expression or production in isolated cells or animalsshowing ALT in the absence of candidate compound(s)). Test compounds orcandidate compounds which are considered positive, i.e., likely to bebeneficial in the treatment of cancer will be those which have asubstantial growth inhibitory effects (e.g., test agents that are ableto reduce the growth of cells preferably by at least 20% more preferablyby at least 50%, and most preferably by at least 80%, still morepreferably by about 90 to 100%.

Such compounds would be important in a number of aspects. They would beimportant in regimens for the treatment of L1RT-related cancers, whetheradministered alone or in combination with chemo- and radiotherapeuticregimens known to one skilled in the art in the treatment of cancer.Alternatively, by simply reducing L1RT, these compounds will beinstrumental in selectively inducing massive apoptosis of cancer cells.

The compounds having the desired pharmacological activity are selectedand may be administered in a physiologically or pharmaceuticallyacceptable carrier to a host for treatment of proliferative diseases,etc. Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered (e.g., nucleic acid, protein,organic compound, a vector or transduced cell), as well as by theparticular method used to administer the composition. Accordingly, thereare a wide variety of suitable formulations of pharmaceuticalcompositions of the present invention.

A pharmaceutical composition in the present invention may containrecombinant products. For example, the antisense oligonucleotides ordsRNA targeted to L1RT can be inserted into any of a number ofwell-known vectors for the transfection of target cells and organisms.For example, nucleic acids are delivered as DNA plasmids, naked nucleicacid, and nucleic acid complexed with a delivery vehicle such as aliposome. Viral vector delivery systems include DNA and RNA viruses(Porter, 2004, Retroviral vectors for suicide gene therapy, Methods MolMed., 90:91-106; Wang et al., 2004, Prolonged and inducible transgeneexpression in the liver using gutless adenovirus: A potential therapyfor liver cancer, Gastroenterology, 126:278-289). In a specificembodiment, a viral vector that contains an antisense L1RT nucleic acidis used. For example, a retroviral vector or adenoviral vector known inthe art for cancer gene therapy can be used. The antisense L1RT nucleicacid to be used in gene therapy is cloned into a suitable vector, whichfacilitates delivery of the gene into a patient.

Methods of non-viral delivery of nucleic acids may include nakedpolynucleotide, agent-enhanced uptake of polynucleotide, microinjection,particle bombardment, liposomes, immunoliposomes, polycation orlipid:nucleic acid conjugates. Delivery can be to cells (ex vivoadministration) or target tissues (in vivo administration) (Narayanan,Antisense therapy of cancer, In Vivo. 1994, 8(5):787-793; Zhang et al.,Anti-oncogene and tumor suppressor gene therapy—examples from a lungcancer animal model, In Vivo. 1994, 8(5):755-769. In a particularembodiment, a nucleic acid molecule is used in which the antisense L1RTsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the antisense L1RT nucleicacid. An example of the sequences that flank 5′ end of L1 RT ORF (ORF2)5′-agaccat caagactagg aagaaactgc atcaactaat gagcaaaatc accagctaacatcata-3′ (SEQ ID NO:7).

The inhibitory or antagonistic agents may be administered in a varietyof ways including orally, topically, parenterally e.g. subcutaneously,intraperitoneally, by viral infection, intravascularly, etc. Dependingupon the manner of introduction, the compounds may be formulated in avariety of ways. Formulations suitable for oral administration can beliquid solutions. Formulations suitable for parenteral administration,such as, for example, by intraarticular (in the joints), intravenous,intramuscular, intradermal, intraperitoneal, and subcutaneous routes,include aqueous and non-aqueous, isotonic sterile injection solutions.In the practice of this invention, compositions can be administered, forexample, by intravenous infusion, orally, topically, parenterally orintraperitoneally. Oral and parenteral administrations are the preferredmethods of administration.

The dose administered to a patient, in the context of the presentinvention, should be sufficient to effect a beneficial therapeuticresponse in the patient over time. The dose will be determined by theefficacy of the particular inhibitor or antagonistic agent employed andthe condition of the patient, as well as the body weight or surface areaof the patient to be treated. The size of the dose also will bedetermined by the existence, nature, and extent of any adverseside-effects that accompany the administration of a particular vector,or transduced cell type in a particular patient.

For administration, compounds of the present invention can beadministered at a rate determined by the LD-50 of the compound, and theside-effects of the compound at various concentrations, as applied tothe mass and overall health of the patient. Administration can beaccomplished via single or divided doses or, e.g., by administration tothe site of a solid tumor in a slow release formulations.

EXAMPLES

The following examples further illustrate the present invention. Theexamples below are carried out using standard techniques, that are wellknown and routine to those of skill in the art, except where otherwisedescribed in detail. The examples are offered by way of illustration andnot by way of limitation.

Example 1 Induction of Telomere Shortening, G2 Arrest and Apoptosis inTelomerase Negative ALT Cells after AZT Treatment

To detect L1 specific RNA in two cell lines (U-2 OS and Saos-2osteosarcomas), reported to maintain telomeres by ALT mechanism⁴, totalmRNA was analyzed by dot blotting with an L1 retrotransposon specificprobe. The reported telomerase-positive cell lines (HEC-1 and HeLa) wereused for comparison^(4,21) (FIG. 1). Both ALT cell lines were positivein this test. HEC-1 cells were completely negative, with only traces ofL1 transcripts in HeLa cells, as previously reported²⁰.

Further to test the proposed method, ALT cell lines were treated withtherapeutic concentrations of AZT, to determine if slippage telomericDNA synthesis could be inhibited by AZT-TP, and thereby induce telomereshortening. Telomere length in AZT treated and untreated cell lines wasmeasured by flow cytometry with a telomere-specific peptide nucleic acid(PNA) probe^(22,23). To determine cell cycle distribution, cells werestained with propidium iodide (PI)²². After 14 days of AZT treatment,both ALT cell lines demonstrated telomere shortening, massive apoptosisand G2 arrest (FIG. 2). To confirm the specificity of AZT-inducedtelomere shortening for ALT cells, a HeLa cell line, known to bepositive for telomerase, was treated with AZT under the same conditions.AZT at the chosen concentration had no effect on telomere length or cellcycle distribution in the HeLa cells (not shown).

To demonstrate telomere shortening and changes in DNA synthesis rate,dynamic, U-2 OS cells were treated with AZT for different amounts oftime, and analyzed by flow cytometry simultaneously. Rate of DNAsynthesis was determined by incorporation of 5-bromodeoxyuridine(BdU)²⁴. Results (FIG. 3) show progressive telomere shortening anddecrease in DNA synthesis. It is important to note that changes in cellcycle distribution, DNA synthesis and telomere length were rapid andcould be detected after only 10 days of AZT treatment.

At the same time, PI staining demonstrated a higher DNA content in AZTtreated cells at later stages of treatment, compared to untreated cells.A rational explanation of this fact is a short telomere inducedchromosome end-to-end joining^(12,26). Induction of apoptosis in AZTtreated ALT cells seems to be p53 independent since U-2 OS and Saos-2represent both p53+/+ and p53−/− cancer cell lines²⁷.

Tumors with suppressed elongation of telomeres have been reported tolose their tumorigenic potential^(12,26), and AZT is already in clinicaluse for treating AIDS. The present disclosure provides that AZT can beused for the treatment of up to 30% of cancer cases. Some othernucleoside reverse transcriptase inhibitors (e.g. 2′,3′-dideoxyinosine(ddI) or 2′,3′-didehydro-3′-deoxythymidine(d4T)) that are already inclinical practice could also be used.

Example 2 Induction of Telomere Shortening, G2 Arrest and Apoptosis inTelomerase Negative ALT Cells after Antisense Inhibition of L1 ReverseTranscriptase

To confirm that ALT is conducted by L1 reverse transcriptase only, U-2OS cells were transfected expressing constructs containing part of humanL1 ORF2 in sense and antisense orientation. The L1 specific reversetranscriptase targeted antisense construct was created as follows: PCRwas performed using RT-F (5′-ATG ACA GGA TCA ACT TCA CAC-3′) (SEQ IDNO:8), RT-R (5′-TCC TGC TTT CTC TTG TAG GCA-3′) (SEQ ID NO:6) primersand pBS-L1RP-EGFP plasmid as a template. 929 bp PCR product was clonedin pTargetT vector (Promega).

Recombinant constructs containing insert in sense and antisenseorientation were purified with Plasmid Midi Kit (Qaigen), digested withXmn I (Promega) and transfected into U-2 OS cells using “Lipofectamine”(Gibco) according to the manufacturers instructions. After 40 days ofselection on media containing 0.5 mg/ml of G418 (Gibco), cells wereharvested, stained with PNA and PI, and analyzed by flow cytometry²². Aschematic representation of L1 reverse transcriptase antisense targetingis shown in FIG. 4.

Data presented in FIG. 5 show that cells carrying antisense constructdemonstrated massive apoptosis, G2 arrest, and telomere shortening asexpected. In contrast, cells expressing sense construct showed nodifference in telomere length or cell cycle.

The following materials and procedures were used in the above workingexamples:

Cell lines: All cell lines used in this study were obtained fromAmerican Type Culture Collection (Rockville, Md.). The cells originsincluded osteosarcoma (Saos-2 and U-2 OS), liver (HEC-1) and uterinecervix (HeLa). Cells were cultured following ATCC recommendation. Fortreatment of the cells with AZT, the media was supplemented with 0.2 μMof 3′-azido-2′,3′-dideoxythymidine (Sigma)²⁸.

Dot blotting: Total cellular RNA was isolated using “RNA-STA 60”solution (Tel-Test, Inc.). The reaction was performed using 30 μg oftotal RNA and “HRP North2South” (Pierce) labeled pBS-L1_(RP)-EGFPplasmid²⁹ as a specific probe, according to the manufacturers protocol.

Bromodeoxyuridine incorporation: Cell staining, for BdU incorporation,was performed using cells which were incubated with 10 mM BrdU (Sigma)for 2.5 h, stained with BU-33 anti-BrdU monoclonal antibodies (Sigma)and FITC labeled Alexa 488 goat anti-mouse IgG (H+L) (Fab′) fragments(Molecular Probes), contrastained with 50 μg/ml PI (Sigma) and analyzedby flow cytometry as described²⁴.

Telomere length measurement by flow cytometry: Cell were stained withtelomere specific FITC conjugated (C₃TA₂)₃ PNA (Applied Biosystems)probe and contrastained with 0.06 μg/ml PI as described.

Inhibition of L1 reverse transcriptase using antisense strategy: Tocreate L1 specific reverse transcriptase targeted antisense constructPCR was performed using RT-F (5′-ATG ACA GGA TCA ACT TCA CAC-3′) (SEQ IDNO:8), RT-R (5′-TCC TGC TTT CTC TTG TAG GCA-3′) (SEQ ID NO:6) primersand pBS-L1_(RP)-EGFP plasmid as a template. 929 bp PCR product wascloned in pTargetT vector (Promega). Recombinant constructs containinginsert in sense and antisense orientation were purified with PlasmidMidi Kit (Qaigen), digested with Xmn I (Promega) and transfected intoU-2 OS cells using “Lipofectamine” (Gibco) according to themanufacturers instructions. After 40 of selection on media containing0.5 mg/ml of G418 (Gibco), cells were harvested, stained with PNA andPI, and analyzed by flow cytometry²².

The references numbered 1-29 below are cited in the above description(with the corresponding superscript numbers) and as such one skilled inthe art would match the references to the appropriate superscriptnumbers in the text above.

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All publications, patents and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this invention pertains. All publications, patents and patentapplications are herein incorporated by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference. Although theforegoing invention has been described in some detail by way ofillustration and example for purposes of clarity of understanding, itwill be obvious that certain changes and modifications may be practicedwithin the scope of the appended claims.

What is claimed is:
 1. A method of selecting a compound capable ofshortening telomeres in telomerase negative cancer cells, the methodcomprising: administering a test compound to said cells; evaluatinganti-L-1 (LINE-1) retrotransposon reverse transcriptase (L1RT) activityof the test compound or evaluating whether the compound down-regulatesexpression of L1RT in said cells; and selecting the compound thatexhibits anti-L-1 retrotransposon activity or down-regulates the L1RTexpression, wherein said cells are either in vitro cultured cells or ina non-human animal model.
 2. The method of claim 1, wherein the step ofevaluating comprises testing for telomere shortening or G2 arrest insaid cells or apoptosis of said cells.
 3. The method of claim 2, whereinsaid cells are in vitro cultured cells.
 4. The method of claim 1,wherein the animal model is selected from the group consisting of amouse, a rat, a rabbit, a pig, a cow, a monkey and a guinea pig.
 5. Amethod of detecting the presence of cancerous cells in a cell samplethat is telomerase negative, the method comprising: contacting saidsample with an inhibitor or antagonist of L-1 (LINE-1) retrotransposonencoded reverse transcriptase (L1RT); and testing for cells exhibitingtelomere shortening or G2 arrest in said cells or apoptosis of saidcells.
 6. A method of detecting cells capable of pathologicallyproliferating in a sample of cells obtained from a tissue of a mammal,comprising contacting the sample of cells with a nucleic acid probe thatis substantially complementary or fully complementary to a subsequenceof an L1RT mRNA, or with an antibody specific to L1RT reversetranscriptase; and detecting L1RT expression in said cells.
 7. Themethod of claim 6, wherein the nucleic acid probe or antisense sequencecomprises a sequence selected from the group consisting of:5′-CCAGAGATTCTGGTATGTGGTGTC TTT GTT-3′ (SEQ ID NO: 2), 5′-CTT TCT CTTGTA GTA GGC ATT TAG TGC TAT AAA-3′ (SEQ ID NO: 3), 5′-CTC TTG CTT TTCTAG TTC TTT TAA TTG TGA-3′ (SEQ ID NO: 4), 5′-CTT CAG TTC TGC TCT GATTTT AGT TAT TTC-3′(SEQ ID NO: 5) and 5′-TCC TGC TTT CTC TTG TAGGCA-3′(SEQ ID NO 6).
 8. The method of claim 7, wherein said nucleosideanalog is 3′-azido-2′,3′dideoxythymidine (AZT), 2′,3′-dideoxyinosine(ddI), 2′3′-didehydro-3′deoxythymidine (d4T), ganciclovir orvalganciclovir, or a combination thereof.
 9. The method of claim 6,wherein the nucleic acid probe comprises a detectable moiety.
 10. Themethod of claim 9, wherein the detectable moiety is a radioisotope, afluorescent molecule, biotin or digioxigenin.
 11. An in vitro method ofinterfering with lengthening of telomeres in telomerase negative tumorcells showing alternative lengthening of telomeres induced or mediatedby L-1 (LINE-1) retrotransposon encoded reverse transcriptase (L1RT),the method comprising administering to the cells an effective amount ofan antisense sequence wherein the antisense sequence hybridizes with anucleic acid sequence encoding the reverse transcriptase and blockslengthening of telomeres in said cells.
 12. The method of claim 11,wherein the L1 nucleic acid sequence is a DNA sequence, an RNAtranscribed from the DNA or a cDNA reverse transcribed from the RNA. 13.The method of claim 11, wherein the antisense sequence comprises achimeric RNA-DNA oligonucleotide.
 14. The method of claim 11, whereinthe antisense sequence comprises a sequence selected from the groupconsisting of: 5′-CCAGAGATTCTGGTATGTGGTGTC TTT GTT-3′ (SEQ ID NO: 2),5′-CTT TCT CTT GTA GGC ATT TAG TGC TAT AAA-3′ (SEQ ID NO: 3), 5′-CTC TTGCTT TTC TAG TTC TTT TAA TTG TGA-3′ (SEQ ID NO: 4), 5′-CTT CAG TTC TGCTCT GAT TTT AGT TAT TTC-3′ (SEQ ID NO: 5) and 5′-TCC TGC TTT CTC TTG TAGGCA-3′ (SEQ ID NO 6).
 15. An in vitro method of inhibiting the growth ofa telomerase negative cell showing alternative lengthening of telomeresinduced or mediated by L-1 (LINE-1) retrotransposon encoded reversetranscriptase (L1RT) in said cell, the method comprising: transfectingthe cell with a construct capable of expressing an antisense sequencethat is fully complementarily to L1RT nucleic acid sequence or to asubsequence of the L1RT nucleic acid sequence in said cell, wherein saidantisense sequence is expressed and the growth of the telomerasenegative cell is inhibited.
 16. The method of claim 15, wherein thenucleic acid is a human L1RT open reading frame.
 17. The method of claim15, wherein the nucleic acid is an L1RT mRNA.
 18. The method of claim15, wherein the antisense sequence is one resulting from the constructexpressing the sequence in SEQ ID NO: 1 in reverse orientation.
 19. Themethod of claim 15, wherein the subsequence of the L1RT nucleic acid isfully complementary to 5′-CCAGAGATTCTGGTATGTGGTGTC TTT GTT-3′ (SEQ IDNO: 2), 5′-CTT TCT CTT GTA GGC ATT TAG TGC TAT AAA-3′ (SEQ ID NO:3),5′-CTC TTG CTT TTC TAG TTC TTT TAA TTG TGA-3′ (SEQ ID NO: 4), 5′-CTT CAGTTC TGC TCT GAT TTT AGT TAT TTC-3′ (SEQ ID NO: 5) or 5′-TCC TGC TTT CTCTTG TAG GCA-3′ (SEQ ID NO 6).
 20. The method of claim 15, wherein theantisense sequence is a DNA oligonucleotide, a 2′-O methyloligonucleotide, a peptide nucleic acid oligonucleotide or aphosphorothioate oligonucleotide.
 21. The method of claim 15, whereinthe antisense L1RT nucleic acid has the nucleotide sequence comprisingSEQ ID NO:
 1. 22. The method of claim 15, wherein the antisense sequenceis about 8 to about 50 nucleotides in length.
 23. The method of claim15, wherein the antisense sequence is about 15 to about 25 nucleotidesin length.
 24. The method of claim 17, wherein the cell is contactedwith two or more antisense sequences fully complementary to differentsubsequences of the nucleic acid.
 25. A composition comprising apolynucleotide capable of encoding a nucleic acid segment capable ofinterfering with L-1 (LINE-1) retrotransposon reverse transcriptase(L1RT) activity in cells.
 26. The composition of claim 25, wherein thenucleic acid segment comprises SEQ ID NO:
 1. 27. An isolated host cellcomprising the composition of a polynucleotide capable of encoding anucleic acid segment capable of interfering with L-1 (LINE-1)retrotransposon reverse transcriptase (L1RT) activity in cells.
 28. Theisolated cell of claim 27, wherein the cell is a human cell.
 29. Theisolated cell of claim 28, wherein the cell is a cancer cell.